| Saliva is one of important human body fluids. Approximately 20% of total salivary proteins are also seen in plasma, and proteins in both fluids show comparable functional diversity and disease-linkage. However, for a subset of diseases, saliva has higher apparent diagnostic potential. These results expand the potential of whole saliva in health monitoring/diagnostics and provide a general platform for improving proteomic coverage of complex biological samples. The analysis of saliva would be a complement to current detection methods due to its advantages such as small transmission risk and noninvasive technique. Salivary concentrations are much lower than those in plasma, so it's difficult to detect by the previous technology. With the rapid development of bio-sensing technologies, the electrochemical immunoassay sensing technology, as a simple, sensitive and selective method, is widely used in the determination of trace substances. Electron transfer between an electrode and the activity center is the basis for developing various electrochemical immunosensor. It can be achieved through two different pathways. On the one hand, the media for electron transfer between an electrode and the activity center is very important; On the other hand, the method and material used to immobilize biomolecules is one of the crucial factors for improving the stability, sensitivity and selectivity of biosensor.In this paper,secretory immunoglobulin A (sIgA), one of the high abundance salivary proteins, was chosen as the detected objects. Nanomaterials with large surface area, high surface free energy, were chosen to immobilize thionine by layer-by-layer(LBL) assembly technique, and the electron mediator was bound onto the electrode surface through electrostatic adsorption with, Au-S and Au-N covalent bonds. For the leakage significantly decrease, the proposed mediator-based salivary electrochemical immunosensor exhibit good analytical performance. In addition, the Nafion/nano-Au (GNPs)composite and multi-wall carbon nanotubes (MWNTs)/nano-Au composite has been paid much attention. The application of these composites as an immobilization matrix for salivary electrochemical immunosensor has been preliminarily investigated. The main points of this dissertation are summarized as follows:1. A novel renewable immunosensor based on Nafion LBL assembly of gold nanoparticles and thionine/sIgA/horseradish peroxidase was developed for the detection of salivary sIgA.A novel amperometric immunosensor has been developed for the detection of salivary sIgA based on LBL assembly of gold nanoparticles and thionine (GNPs-THI) on the Nafion modified glassy carbon electrode (GCE) and the sIgA was adsorbed by the formative monolayer GNPs, nonspecific adsorption sites were blocked by horseradish peroxidase (HRP) eventually. The immunosensor showed high sensitivity and specificity for sIgA with a detection limit of 3.0 ng/mL and the linear range was from 6.5 to 300 ng/mL. And the response time achieved 95% steady state was less than 20 s. Some influencing factors to the biosensor were investigated, such as the concentration of antibody and substrate, pH and temperature, incubation time, interferents. To detect salivary samples parallel using the method of biosensor and ELISA, the results indicated that the two methods had significant correlation(r=0.98932, P<0.001), so it can be used to detect salivary sIgA fast to judge the localimmunity of human body.2. A novel renewable immunosensor based on functional multi-wall carbon nanotubes/LBL assembly of gold nanoparticles and polythionine/sIgA/horseradish peroxidase was developed for the detection of salivary sIgA.A novel amperometric immunosensor based on MWNTs/ LBL gold nanoparticles and polythionine (GNPs-PTH)/ sIgA/ HRP was developed for the detection of salivary sIgA. The MWNTs film distributed by chitosan was produced a negatively charged interface. The LBL assembly of GNPs-PTH was assembled through electrostatic adsorption with, Au-S and Au-N covalent bonds. Then the salivary anti-sIgA antibody was adsorbed by the formative monolayer of GNPs. Eventually, the HRP was used to block the nonspecific adsorption sites on the modified GCE and amplify the current signal at the same time. The immunosensor showed high sensitive to salivary sIgA with a detection limit(S/N=3) of 1.5μg/L and the linear range was 3.0-350.0μg/L, with correlation coefficient (r) of 0.9897 (P<0.001). The response time achieved 95% of the steady state was less than 20 s. Also, the immunosensor had an excellent antiturbulence for the interferents of immunoglobulin G (IgG),α-amylase and albumin, and the interference rates were -2.13%, -2.85% and -1.96% respectively. The antigen-antibody complexes in the measuring were decollemented by 0.1 mol/L of aminoacetic acid. The bioactivities of antigen and antibody were remained well after decollement and the biosensor could be recycled by aminoacetic acid for eight times. Some influencing factors to the biosensor were investigated, such as the concentration of substrate and antibody, incubation time, interferents, temperature and pH. It can be used for accurate and fast detection of salivary sIgA to judge the impairment of the mucosal immune system of human owing to its high sensitivity, excellent specificity, convenient detection and it can be easily regenerated.
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